Reaction products of alkenyl dihydrofuran-2,5-dione and polyisoalkenyl dihydro-2,5-furandione with alkylene glycol

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
See read-across justification attached in Section 13 of the IUCLID.

Analytical monitoring:

not required

Details on sampling:

The rate of respiration was measured over an approximately 10 minute period at 0 hours and after 3 hours contact time.

Details on test solutions:

The test concentration to be used in the definitive test was determined by a preliminary range-finding test.
In the range-finding test activated sewage sludge micro-organisms were exposed to nominal test concentrations of 1.0, 10, 100 and 1000 mg/L. The test material was dispersed directly in water.
An amount of test material (4.00 g) was dispersed in water with teh aid of ultrasonication and the volume adjsted to 1L to give a 4.0g/L stock solution. A dilution was made from this stock solution to give a 40 mg/L stock solution. Aliquots (12.5 and 125 mL) of the 40 mg/L stock solution were removed and dispersed with activated sewage sludge, synthetic sludge, synthetic sewage and water to give the test concentrations of 1.0 and 10 mg/L. Similarly aliquots (12.5 and 125 mL) of the 4.0 g/L stock solution were used to prepare the 100 and 1000 mg/L test concentrations.
Synthetic sewage (16 mL) diluted to 300 mL with water and activated sewage sludge (200 mL) and water were added to the test vessels and the mixture aerated. The precudure was repeated every 15 minutes with appropriate amounts of test or reference material being added..
A reference material 3,5-dichlorophenol was included in the range-finding test at concentrations of 3.2 and 32 mg/L in order to confirm the suitability of the inoculum.
Based on the results of the range-finding study, the following test concentrations were assigned to the definitive study: 100, 180, 320, 560 and 1000 mg/L. The required amount of test material was added to each test vessel using the method described above.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Test Species
A mixed population of activated sewage sludge micro-organisms was obtained on 23 August 1995 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK which treats predominantly domestic sewage.

Synthetic Sewage
A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:
16 g Peptone
11 g Meat extract
3 g Urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
dissolved in 1 litre of water with the aid of ultrasonication.

Preparation of inoculum
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and was fed 50 mL/L of synthetic sewage sludge prior to use. The pH of the sample on the day of the test was 7.3 and the suspended solids equal to 3.9 g/L prior to use.

Test type:

static

Water media type:

freshwater

Total exposure duration:

3 h

Post exposure observation period:

Not applicable.

Hardness:

100 mg/L CaCO3

Test temperature:

21°C

pH:

not measured.

Dissolved oxygen:

1.5 - 8.6 mg O2/L

Salinity:

Not applicable

Nominal and measured concentrations:

Range finder: 1.0, 10, 100 and 1000 mg/L.
Definitive: 100, 180, 320, 560 and 1000 mg/L

Details on test conditions:

Preparation of test system
At time "0" 16 ml of synthetic sewage was diluted to 300 mL with water and 200 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with compressed air via narrow bore glass tubes at a rate of approximately 0.5 – 1 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the test or reference item being added. Finally a second control vessel was prepared.
As each vessel reached 3 hours contact time the contents of the conical flask were poured into the measuring vessel (250 ml darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over an approximate 10 minute period (between approximately 8.6 mg O2/l and 1.5 mg O2/l). This procedure was repeated after 3 hours contact time.
The test was conducted under normal laboratory lighting in a temperature controlled room at 21ºC.

Evaluation of Data
The percentage inhibition was calculated according to the equation:
% inhibition = [1 – (2RS/RC1 + RC2)] x 100
Where:
RS = oxygen consumption rate for test or reference sample
RC1 + RC2 = oxygen consumption rates for controls 1 and 2
The percentage inhibition values were plotted against concentration and the EC50 values derived by inspection of the graph..

The results of the study are considered valid if (i) the two control respiration rates are within 15% of each other and (ii) the EC50 (3-Hour contact time) for 3,5-dichlorophenol lies within the range 5 to 30 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

390 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other: No confidence limits stated

Details on results:

Range-finder
Significant inhibition of respiration was observed at 1000 mg/L, however, there were no effects on respiration at 1.0, 10 and 100 mg/L. Based on this information test concentrations of 100, 180, 320, 560 and 1000 mg/L were selected for the main study.

Definitive study
EC 50 (30 mins) > 1000 mg/L
EC50 (3h) = 390 mg/L
Variation in respiration rates of controls 1 and 2 after 3 hours: 0%
The validation criteria for the control respiration rates and reference material EC50 values have been satisfied.

See attached document for result tables and graphs.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50 (3 h) = 7.0 mg/L

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test material to microorganisms was assessed in accordance with OECD Guideline 209. The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of 390 mg/L.

Executive summary:

Introduction.

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test" and EC Commission Directive 87/302/EEC.

Methods.

 The test material was aerated for a period of 3 hours at a temperature of 21°C in the presence of activated sludge with synthetic sewage as a respiratory substrate. The rate of respiration was determined after 30 minutes and 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

Results and Conclusion.

The effect of the test item on the respiration of activated sewage sludge gave a 3‑Hour EC₅₀value of 390 mg/L.

The reference item gave a 3-Hour EC₅₀value of 7.0 mg/L.

Description of key information

The toxicity of the test material to microorganisms was assessed in accordance with OECD Guideline 209.  The effect of the test item on the respiration of activated sewage sludge gave a 3‑Hour EC50 value of 390 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

390 mg/L

Additional information

The key study was performed on the read-across substance Ethylene glycol, reaction products with polyisobutenyl succinic anhydride and hexadecenyl succinic anhydride, salts with dimethylethanolamine. The justification for read-across is attached in Section 13 of the IUCLID.

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test" and EC Commission Directive 87/302/EEC.

 The test material was aeratedfor a period of 3 hours at a temperature of 21°C in the presence of activated sludge with synthetic sewage as a respiratory substrate. The rate of respiration was determined after 30 minutes and 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

The effect of the test item on the respiration of activated sewage sludge gave a 3‑Hour EC₅₀value of 390 mg/L.

The reference item gave a 3-Hour EC₅₀value of 7.0 mg/L